Abundance Of Membrane Proteins Research Articles

Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions.

Campylobacter jejuni is responsible for the major foodborne bacterial enteritis in humans. In contradiction with its fastidious growth requirements, this microaerobic pathogen can survive in aerobic food environments, suggesting that it must employ a variety of protection mechanisms to resist oxidative stress. For the first time, C. jejuni 81–176 inner and outer membrane subproteomes were analyzed separately using two-dimensional protein electrophoresis (2-DE) of oxygen-acclimated cells and microaerobically grown cells. LC-MS/MS analyses successfully identified 42 and 25 spots which exhibited a significantly altered abundance in the IMP-enriched fraction and in the OMP-enriched fraction, respectively, in response to oxidative conditions. These spots corresponded to 38 membrane proteins that could be grouped into different functional classes: (i) transporters, (ii) chaperones, (iii) fatty acid metabolism, (iv) adhesion/virulence and (v) other metabolisms. Some of these proteins were up-regulated at the transcriptional level in oxygen-acclimated cells as confirmed by qRT-PCR. Downstream analyses revealed that adhesion of C. jejuni to inert surfaces and swarming motility were enhanced in oxygen-acclimated cells or paraquat-stressed cells, which could be explained by the higher abundance of membrane proteins involved in adhesion and biofilm formation. The virulence factor CadF, over-expressed in the outer membrane of oxygen-acclimated cells, contributes to the complex process of C. jejuni adhesion to inert surfaces as revealed by a reduction in the capability of C. jejuni 81–176 ΔCadF cells compared to the isogenic strain.Taken together, these data demonstrate that oxygen-enriched conditions promote the over-expression of membrane proteins involved in both the biofilm initiation and virulence of C. jejuni.

Regulation of ecto-5′-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors

Ecto-5′-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several in vivo models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H2O2) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H2O2 decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.

Temperature‐dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion

Human limb muscle and skin blood flow increases significantly with elevations in temperature, possibly through physiological processes that involve temperature-sensitive regulatory mechanisms. Here we tested the hypothesis that the release of the vasodilator ATP from human erythrocytes is sensitive to physiological increases in temperature both in vitro and in vivo, and examined potential channel/transporters involved. To investigate the source of ATP release, whole blood, red blood cells (RBCs), plasma and serum were heated in vitro to 33, 36, 39 and 42°C. In vitro heating augmented plasma or ‘bathing solution’ ATP in whole blood and RBC samples, but not in either isolated plasma or serum samples. Heat-induced ATP release was blocked by niflumic acid and glibenclamide, but was not affected by inhibitors of nucleoside transport or anion exchange. Heating blood to 42°C enhanced (P < 0.05) membrane protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in RBCs. In a parallel in vivo study in humans exposed to whole-body heating at rest and during exercise, increases in muscle temperature from 35 to 40°C correlated strongly with elevations in arterial plasma ATP (r2 = 0.91; P = 0.0001), but not with femoral venous plasma ATP (r2 = 0.61; P = 0.14). In vitro, however, the increase in ATP release from RBCs was similar in arterial and venous samples heated to 39°C. Our findings demonstrate that erythrocyte ATP release is sensitive to physiological increases in temperature, possibly via activation of CFTR-like channels, and suggest that temperature-dependent release of ATP from erythrocytes might be an important mechanism regulating human limb muscle and skin perfusion in conditions that alter blood and tissue temperature.

Plant potassium content modifies the effects of arbuscular mycorrhizal symbiosis on root hydraulic properties in maize plants

It is well known that the arbuscular mycorrhizal (AM) symbiosis helps the host plant to overcome several abiotic stresses including drought. One of the mechanisms for this drought tolerance enhancement is the higher water uptake capacity of the mycorrhizal plants. However, the effects of the AM symbiosis on processes regulating root hydraulic properties of the host plant, such as root hydraulic conductivity and plasma membrane aquaporin gene expression, and protein abundance, are not well defined. Since it is known that K(+) status is modified by AM and that it regulates root hydraulic properties, it has been tested how plant K(+) status could modify the effects of the symbiosis on root hydraulic conductivity and plasma membrane aquaporin gene expression and protein abundance, using maize (Zea mays L.) plants and Glomus intraradices as a model. It was observed that the supply of extra K(+) increased root hydraulic conductivity only in AM plants. Also, the different pattern of plasma membrane aquaporin gene expression and protein abundance between AM and non-AM plants changed with the application of extra K(+). Thus, plant K(+) status could be one of the causes of the different observed effects of the AM symbiosis on root hydraulic properties. The present study also highlights the critical importance of AM fungal aquaporins in regulating root hydraulic properties of the host plant.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

AbstractPlatelets are activated on increase of cytosolic Ca2+ activity ([Ca2+]i), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+]i increase are significantly blunted in platelets from SGK1 knockout mice (sgk1−/−). Similarly, Ca2+-dependent degranulation, integrin αIIbβ3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1−/− platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1−/− mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active S422DSGK1 but not with inactive K127NSGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with S422DSGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of S422DSGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB–dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.

Nucleotide Sequence of the Na+/H+ Exchanger‐8 in Patients With Congenital Sodium Diarrhea

Sodium absorption by the intestine is mediated by brush border Na/H exchangers, which include the NHE3 and NHE8 isoforms. We demonstrated a maturational decrease in NHE8 and increase in NHE3 in mouse intestine mRNA abundance and brush border membrane protein abundance, indicating a developmental switch of isoforms. Congenital sodium diarrhea is a rare autosomal recessive disorder characterized by polyhydramnios, hyponatremia, metabolic acidosis, and diarrhea with a high sodium content. Previous studies using intestinal brush border membrane vesicles from patients with this disorder have demonstrated a decrease in Na/H exchanger activity. Because some patients with congenital sodium diarrhea improve with age and knowing the developmental switch from NHE8 to NHE3, NHE8 may be a candidate gene for this disorder. We sequenced NHE8 from 5 patients with this disorder and found no disease-causing homozygous mutations. Although brush border membrane Na/H exchange activity may be decreased, exonic mutations in NHE8 cannot account for this disorder in these subjects.

Rapid Response of the Yeast Plasma Membrane Proteome to Salt Stress

The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88-109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H(+)-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place.

Challenges in plasma membrane phosphoproteomics

The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.

Analysis of Affinity Maps of Membrane Proteins on Individual Human Embryonic Stem Cells

The heterogeneity found in many cell types has greatly inspired research in single-cell gene and protein profiling for discovering the origin of heterogeneity and its role in cell fate decisions. Among the existing techniques to probe heterogeneity, atomic force microscopy (AFM) utilizes an antibody/ligand-modified tip to explore the distribution of a target membrane protein on individual cells in their native environment. In this paper, we establish a practical model to analyze the data systematically, and attempt the quantification of membrane protein abundance on single cells by taking account issues, such as the level of nonspecific interaction, the probe resolution, and the reproducibility of detecting protein distribution. We demonstrated the application in examining the heterogeneous distribution and the local protein abundance of TRA-1-81 antigen on human embryonic stem (hES) cells at the subcellular level. Heterogeneity in TRA-1-81 expression was also detected at the single cell level, suggesting the presence of subpopulation cells within an undifferentiated hES cell colony. The method provides a platform to unveiling the correlation between heterogeneity of membrane proteins and cell development in a complex cell community.

The Virulence Factor PEB4 (Cj0596) and the Periplasmic Protein Cj1289 Are Two Structurally Related SurA-like Chaperones in the Human Pathogen Campylobacter jejuni

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, Δ G°(H2O), for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

Effect of dexamethasone on Na+/Ca2+ exchanger in dendritic cells

Ca(+)-dependent signaling regulates the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. The activity of DCs is suppressed by glucocorticoids, potent immunosuppressive hormones. The present study explored whether the glucocorticoid dexamethasone influences the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in DCs. To this end, DCs were isolated from mouse bone marrow. According to fura-2 fluorescence, exposure of DCs to lipopolysaccharide (LPS, 100 ng/ml) increased [Ca(2+)](i), an effect significantly blunted by overnight incubation with 10 nM dexamethasone before LPS treatment. Dexamethasone did not affect the Ca(2+) content of intracellular stores, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 and SERCA3 expression, ryanodine receptor (RyR)1 expression, or Ca(2+) entry through store-operated Ca(2+) channels. In contrast, dexamethasone increased the transcript level and the membrane protein abundance of the Na(+)/Ca(2+) exchanger NCX3. The activity of Na(+)/Ca(2+) exchangers was assessed by removal of extracellular Na(+) in the presence of external Ca(2+), a maneuver triggering the Ca(2+) influx mode. Indeed, Na(+) removal resulted in a rapid transient increase of [Ca(2+)](i) and induced an outwardly directed current as measured in whole cell patch-clamp experiments. Dexamethasone significantly augmented the increase of [Ca(2+)](i) and the outward current following removal of extracellular Na(+). The NCX blocker KB-R7943 reversed the inhibitory effect of dexamethasone on LPS-induced increase in [Ca(2+)](i). Dexamethasone blunted LPS-induced stimulation of CD86 expression and TNF-α production, an effect significantly less pronounced in the presence of NCX blocker KB-R7943. In conclusion, our results show that glucocorticoid treatment blunts LPS-induced increase in [Ca(2+)](i) in DCs by increasing expression and activity of Na(+)/Ca(2+) exchanger NCX3. The effect contributes to the inhibitory effect of the glucocorticoid on DC maturation.

Focus on Physical Principles of Protein Behavior in the Cell

What are the principles that regulate the complex network of biochemical interactions in a living cell? To address this question, it is of central importance to understand the organization of the proteome, as proteins are involved in essentially all the processes that take place in the cell, including those required for the control of gene expression and metabolism. We are witnessing an explosion of novel experimental high-throughput techniques and theoretical analysis methods to elucidate the mechanisms of protein regulation, localization, and interaction. The physical principles underlying these advances are the subject of this Focus Issue of PROTEOMICS. The Focus Issue has been developed from an interdisciplinary conference that took place at the Max Planck Institute for the Physics of Complex Systems in Dresden, Germany, October 26–30, 2009. The event brought together researchers from all major areas relevant to understanding protein behavior in the cell, including protein expression, localization, function, folding, misfolding, and aggregation. The present Focus Issue brings together a selection of papers where opinion leaders in the field discuss the manner in which existing concepts and methods have made it possible to carry out exciting new studies of the networks of interacting molecules that underlie the myriad biological pathways through which cells maintain homeostasis and promote development. There is increasing expectation that in-depth research addressing the physical principles that underlie protein behavior in the cell will greatly improve existing methods for characterizing and predicting structures, interactions, and functions of proteins, for modeling their evolution, and ultimately for suggesting new rational approaches for treating human diseases. The topics covered in this Focus Issue are quite broad, ranging from experimental studies to computer modeling. Müller and co-workers investigate the energy landscape in the unfolding reaction of the E. coli outer membrane protein G using dynamic single-molecule force spectroscopy and relate it to functional parameters of the protein such as rigidity and flexibility. Wilke and collaborators study the association between optimal codon usage and protein structural properties in the genomes of four model organisms and show that aggregation propensity and solvent accessibility have independent effects on codon usage. The relationship between stability and folding properties of a protein sequence in vitro and in situ with its aggregation properties in vivo is studied by Ventura and co-workers using SH3 domains as example. This paper is followed by one by Schroeder and collaborators describing the structural modeling of the histone methyltransferase complex Set1C in yeast, which consists of eight subunits, through homology modeling and computational docking. Then, Schwille and her collaborators investigate the relative abundance of bitopic membrane proteins and the amino acid composition of their transmembrane domains in different taxa and determine how the context of the transmembrane domains of bitopic receptors function in the assembly of dimers, whereas Marcotte and his group study the conservation of mRNA and protein abundances comparing seven different species, showing that protein abundances are generally more conserved than mRNA abundances. The Focus Issue is concluded by a paper by Lilley and collaborators who describe a refined bioinformatic approach to assign proteins of uncharacterized localization to subcellular structures. The Editors wish to thank the Editor-in-Chief of PROTEOMICS, Professor Mike Dunn, the Managing Editor, Dr. Hans Joachim Kraus, and the publisher Wiley-VCH for offering them the opportunity to prepare this Focus Issue. They wish to warmly thank all the authors for exciting contributions for the issue, and the reviewers for their critical and highly qualified evaluation of the contributions. The Editors would also like to thank the Max Planck Institute for the Physics of Complex Systems for financing and hosting the interdisciplinary conference which formed the starting point for the Focus Issue. In particular they would like to thank Dr. Sergej Flach (head of the conference program) for his very helpful support and Mrs. Claudia Pönisch (conference secretary) for her very efficient organization. Markus Porto Cologne H. Eduardo Roman Dresden Michele Vendruscolo Cambridge

Sec23b deficiency In Mice Results In Pancreatic Destruction and Defective long Term Hematopoietic Stem Cell Function

AbstractAbstract 2038Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disorder caused by mutations in the gene SEC23B. SEC23B is a component of the COPII coat complex that transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. CDAII is characterized by mild to moderate anemia and >10% bi- or multi- nucleate erythroblasts in the bone marrow. We generated Sec23b deficient mice (Sec23b gt/gt) from ES cells with a genetrap cassette inserted into the last intron of Sec23b. Sec23b gt/gt mice die at birth with destruction of the exocrine pancreas. Eight to 12 week old lethally irradiated C57BL/6J recipients were transplanted with 106 E17.5 fetal liver cells from either wildtype or Sec23b gt/gt mice. Mice were bled at 6, 8 and 12 weeks post transplant and no significant difference was observed in either hematocrit or hemoglobin (N=3-7 mice per group). Red blood cell (rbc) ghosts prepared from peripheral blood of transplanted mice showed no shift of Band 3 on SDS-PAGE analysis. Ghost fractions from Sec23b gt/gt recipients analyzed by western blot for the presence of residual ER proteins showed no difference between wildtype and Sec23b gt/gt fetal liver cell recipients. No significant difference was found in bi-nucleate erythroblasts in the bone marrow, myeloid:erythroid ratio or spleen mass. We quantitatively analyzed the proteome of mature rbc ghosts from Sec23b gt/gt recipients by Stabile Isotope Labeling of Amino acids in Cells (SILAC) proteomics using rbc ghosts from mice fed lysine labeled with 13C (SILAC chow). When SILAC-labeled rbc ghosts were compared by mass spectrometry analysis with ghosts from Sec23b gt/gt fetal liver cell recipients, no significant differences in abundance of rbc membrane proteins were observed. To more stringently test the ability of Sec23b gt/gt fetal liver cells to engraft, 5×105 wildtype or Sec23b gt/gt fetal liver cells were co-transplanted with 5×105 wildtype fetal liver cells expressing a GFP transgene. FACS analysis of peripheral blood obtained at 6 and 8 weeks post transplant showed significant out-competition of Sec23b gt/gt cells by GFP+ progenitors in Ter119+ rbcs (p<0.01, at all time points), CD3+ T cells (p<0.01 at 8 weeks), B220+ B cells (p<0.01, at all time points) and Mac1+Gr1+ myeloid cells (p<0.01, at all time points) compared to wildtype controls (N=5-7 mice per group). Fetal livers from wildtype and Sec23b gt/gt mice showed no difference in the total number of CD150+CD48-Sca1+cKit+(lineage-) long term hematopoietic stem cells (LT-HSCs) per fetal liver (p=0.45). In conclusion, Sec23b deficient humans and mice exhibit disparate phenotypes, apparently restricted to CDAII in humans and a prominent neonatal pancreatic insufficiency in mice. These differences could be due to evolutionary changes in relative expression and/or function of the Sec23a and b paralogs in humans and mice. However, we cannot exclude an allele-specific defect due to residual or aberrant function of the Sec23b gt allele. Analyses of a deleted and a conditional floxed Sec23b null allele are currently in progress. Finally our transplant data suggest that Sec23b traffics cargoes important for the function of murine LT-HSCs. This finding contrasts with the apparently erythroid-specific human phenotype, raising the possibility of a requirement for Sec23b function in the marrow microenvironment not tested by these transplant studies. Tissue specific deletion of Sec23b is currently in progress to address this question. Disclosures:No relevant conflicts of interest to declare.

Effect of metabolic acidosis on neonatal proximal tubule acidification

The serum bicarbonate in neonates is lower than adults due in large part to a lower rate of proximal tubule acidification. It is unclear if the neonatal proximal tubule is functioning at maximal capacity or if the proximal tubule can respond to metabolic acidosis as has been described in adult proximal tubules. We find that neonatal mouse brush-border membranes have a lower Na(+)/H(+) exchanger (NHE) 3 protein abundance (neonate 0.11 ± 0.05 vs. adult 0.64 ± 0.07; P < 0.05) and a higher NHE8 protein abundance (neonate 1.0 ± 0.01 vs. adult 0.13 ± 0.09; P < 0.001) compared with adults. To examine if neonates can adapt to acidosis, neonatal mice were gavaged with either acid or vehicle for 4 days, resulting in a drop in serum bicarbonate from 19.5 ± 1.0 to 8.9 ± 0.6 meq/l (P < 0.001). Proximal convoluted tubule Na(+)/H(+) exchanger activity (dpH(i)/dt) was 1.68 ± 0.19 pH units/min in control tubules and 2.49 ± 0.60 pH units/min in acidemic neonatal mice (P < 0.05), indicating that the neonatal proximal tubule can respond to metabolic acidosis with an increase in Na(+)/H(+) exchanger activity. Similarly, brush-border membrane vesicles from neonatal rats had an increase in Na(+)/H(+) exchanger activity with acidemia that was almost totally inhibited by 10(-6) M 5-(N-ethyl-n-isopropyl)-amiloride, a dose that has little effect on NHE3 but inhibits NHE8. There was a significant increase in both NHE3 (vehicle 0.35 ± 0.07 vs. acid 0.73 ± 0.07; P < 0.003) and NHE8 brush-border membrane protein abundance (vehicle 0.41 ± 0.05 vs. acid 0.73 ± 0.06; P < 0.001) in acidemic mouse neonates compared with controls. A comparable increase in NHE3 and NHE8 was found in neonatal rats with acidosis. In conclusion, the neonatal proximal tubule can adapt to metabolic acidosis with an increase in Na(+)/H(+) exchanger activity.

Single nucleotide polymorphisms in the human Na+-dicarboxylate cotransporter affect transport activity and protein expression

The sodium-coupled transport of citric acid cycle intermediates in the intestine and kidney is mediated by the Na(+)-dicarboxylate cotransporter, NaDC1. In the kidney, NaDC1 plays an important role in regulating succinate and citrate concentrations in the urine, which may have physiological consequences including the development of kidney stones. In the present study, the impact of nonsynonymous single nucleotide polymorphisms (SNPs) on NaDC1 expression and function was characterized using the COS-7 cell heterologous expression system. The I550V variant had an increased sensitivity to lithium inhibition although there were no significant effects on protein abundance. The L44F variant had no significant effects on expression or function. The membrane protein abundance of the M45L, V117I, and F254L variants was decreased, with corresponding decreases in transport activity. The A310P variant had decreased protein abundance as well as a change in substrate selectivity. The P385S variant had a large decrease in succinate transport V(max), as well as altered substrate selectivity, and a change in the protein glycosylation pattern. The most damaging variant was V477M, which had decreased affinity for both succinate and sodium. The V477M variant also exhibited stimulation by lithium, indicating a change in the high-affinity cation binding site. We conclude that most of the naturally occurring nonsynonymous SNPs affect protein processing of NaDC1, and several also affect functional properties. All of these mutations are predicted to decrease transport activity in vivo, which would result in decreased intestinal and renal absorption of citric acid cycle intermediates.

Changing the phospholipid composition of Staphylococcus aureus causes distinct changes in membrane proteome and membrane‐sensory regulators

The dynamic lipid composition of bacterial cytoplasmic membranes has a profound impact on vital bacterial fitness and susceptibility to membrane-damaging agents, temperature, or osmotic stress. However, it has remained largely unknown how changes in lipid patterns affect the abundance and expression of membrane proteins. Using recently developed gel-free proteomics technology, we explored the membrane proteome of the important human pathogen Staphylococcus aureus in the presence or absence of the cationic phospholipid lysyl-phosphatidylglycerol (Lys-PG). We were able to detect almost half of all theoretical integral membrane proteins and could reliably quantify more than 35% of them. It is worth noting that the deletion of the Lys-PG synthase MprF did not lead to a massive alteration but a very distinct up- or down-regulation of only 1.5 or 3.5% of the quantified proteins. Lys-PG deficiency had no major impact on the abundance of lipid-biosynthetic enzymes but significantly affected the amounts of the cell envelope stress-sensing regulatory proteins such as SaeS and MsrR, and of the SaeS-regulated proteins Sbi, Efb, and SaeP. These data indicate very critical interactions of membrane-sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity.

The Pollen Organelle Membrane Proteome Reveals Highly Spatial−Temporal Dynamics during Germination and Tube Growth of Lily Pollen

As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain.